A dynamic template complex mediates Munc18-chaperoned SNARE assembly.

Munc18 chaperones assembly of three membrane-anchored soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a four-helix bundle to mediate membrane fusion between vesicles and plasma membranes, leading to neurotransmitter or insulin release, glucose transporter (GLUT4) translocation, or other exocytotic processes. Yet, the molecular mechanism underlying chaperoned SNARE assembly is not well understood. Recent evidence suggests that Munc18-1 and Munc18-3 simultaneously bind their cognate SNAREs to form ternary template complexes - Munc18-1:Syntaxin-1:VAMP2 for synaptic vesicle fusion and Munc18-3:Syntaxin-4:VAMP2 for GLUT4 translocation and insulin release, which facilitate the binding of SNAP-25 or SNAP-23 to conclude SNARE assembly. Here, we further investigate the structure, dynamics, and function of the template complexes using optical tweezers. Our results suggest that the synaptic template complex transitions to an activated state with a rate of 0.054 s-1 for efficient SNAP-25 binding. The transition depends upon the linker region of syntaxin-1 upstream of its helical bundle-forming SNARE motif. In addition, the template complex is stabilized by a poorly characterized disordered loop region in Munc18-1. While the synaptic template complex efficiently binds both SNAP-25 and SNAP-23, the GLUT4 template complex strongly favors SNAP-23 over SNAP-25, despite the similar stabilities of their assembled SNARE bundles. Together, our data demonstrate that a highly dynamic template complex mediates efficient and specific SNARE assembly.

Megaviruses contain various genes encoding for eukaryotic vesicle trafficking factors.

Many intracellular pathogens, such as bacteria and large viruses, enter eukaryotic cells via phagocytosis, then replicate and proliferate inside the host. To avoid degradation in the phagosomes, they have developed strategies to modify vesicle trafficking. Although several strategies of bacteria have been characterized, it is not clear whether viruses also interfere with the vesicle trafficking of the host. Recently, we came across SNARE proteins encoded in the genomes of several bacteria of the order Legionellales. These pathogenic bacteria may use SNAREs to interfere with vesicle trafficking, since SNARE proteins are the core machinery for vesicle fusion during transport. They assemble into membrane-bridging SNARE complexes that bring membranes together. We now have also discovered SNARE proteins in the genomes of diverse giant viruses. Our biochemical experiments showed that these proteins are able to form SNARE complexes. We also found other key trafficking factors that work together with SNAREs such as NSF, SM, and Rab proteins encoded in the genomes of giant viruses, suggesting that viruses can make use of a large genetic repertoire of trafficking factors. Most giant viruses possess different collections, suggesting that these factors entered the viral genome multiple times. In the future, the molecular role of these factors during viral infection need to be studied.

Single-Molecule Manipulation Study of Chaperoned SNARE Folding and Assembly with Optical Tweezers.

Intracellular membrane fusion is primarily driven by coupled folding and assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE assembly is intrinsically inefficient and must be chaperoned by a family of evolutionarily and structurally conserved Sec1/Munc-18 (SM) proteins. The physiological pathway of the chaperoned SNARE assembly has not been well understood, partly due to the difficulty in dissecting the many intermediates and pathways of SNARE assembly and measure their associated energetics and kinetics. Optical tweezers have proven to be a powerful tool to characterize the intermediates involved in the chaperoned SNARE assembly. Here, we demonstrate the application of optical tweezers combined with a homemade microfluidic system into studies of synaptic SNARE assembly chaperoned by their cognate SM protein Munc18-1. Three synaptic SNAREs and Munc18-1 constitute the core machinery for synaptic vesicle fusion involved in neurotransmitter release. Many other proteins further regulate the core machinery to enable fusion at the right time and location. The methods described here can be applied to other proteins that regulate SNARE assembly to control membrane fusion involved in numerous biological and physiological processes.